Rapid, tunable, and multiplexed detection of RNA using convective array PCR

Detection of RNA targets is typically achieved through RT-qPCR or RNAseq. RT-qPCR is rapid but limited in number and complexity of targets detected, while RNAseq is high-throughput but takes multiple days. We demonstrate simultaneous amplification and detection of 28 distinct RNA targets from a single unsplit purified RNA sample in under 40 minutes using our convective array PCR (caPCR) technology. We integrate tunable strand displacement probes into caPCR to allow detection of RNA species with programmable sequence selectivity for either a single, perfectly matched target sequence or for targets with up to 2 single-nucleotide variants within the probe-binding regions. Tunable probes allow for robust detection of desired RNA species against high homology background sequences and robust detection of RNA species with significant sequence diversity due to community-acquired mutations. As a proof-of-concept, we experimentally demonstrated detection of 7 human coronaviruses and 7 key variants of concern of SARS-CoV-2 in a single assay.

precipitation solution + 30 μL nuclease-free water) including 30-minute precipitation at -20 °C, centrifugation for 15 minutes at °C, ethanol wash, drying, and elution in 100 μL nuclease-free water.Concentrations of final RNA stocks were quantified using a DeNovix DS-11 Fluorometer/Spectrophotometer.Surface functionalization.Attachment of the pre-annealed probe to the surface of the fluidic chamber is performed via strain-promoted azide-alkyne cycloaddition between the dibenzocyclooctyne-functionalized probe and the azide (N3)-functionalized surface of the fluidic chamber.N3 surface functionalization is performed according to the following protocol.Step 1: glass slides are treated with sodium hydroxide solution (10 M) for 30min in Branson ultrasonic bath (Bransonic CPXH Digital Bath 2800) at the high power of ultrasound generation.
Step 2: After washing with copious amounts of water and drying at 120 °C, the slides are treated with oxygen plasma for 30 min in a Harrick plasma cleaner at high power and maintaining oxygen pressure in a range of 0.4-0.6 torr.Step 3: hydroxylated slides are treated with a 5% solution of N-(2-aminoethyl)-2,2,4-trimethyl-1-azasila cyclopentane (Gelest) in dry dichloromethane (Sigma) for 16 h at room temperature, followed by washing with 95% ethanol in water solution; amino-silanized slides are baked in ambient atmosphere at 105 °C for 2 h.Step 4: functionalization of the amino-terminated slides with an isothiocyanate moiety is performed with 10 mM solution of p-phenylene diisothiocyanate (Sigma) in dry pyridine-dimethylformamide (both chemicals are from Sigma) mixture (9:1, v/v) for 2 h at room temperature, followed by washing with 95% ethanol in water solution and drying at 80 °C for 1 h.Step 5: azide functionalization of the isothiocyanate slides is performed at 37 °C for 2 h with a solution of 11azido-3,6,9-trioxaundecan-1-amine (50 µl) in 0.1 M phosphate buffer pH 8 (10 mL), followed by washing with copious amount of water and drying at 45 °C.At this stage, the slides are ready for microarray printing or can be stored vacuum-sealed at 4 °C for at least 6 months without loss of conjugation activity.
For data shown in Figure 3 and Figure 5, arrays were spotted onto COP rather than glass using a broadly similar method to that described above but lacking the NaOH step and with a shortened plasma treatment (10 minutes).
Probe annealing.Toehold microarray probes were annealed in 2× PBS buffer according to the following protocol: 90 °C for 3 min, then uniformly decreasing temperature down to 30 °C over a period of 1 h.Concentrations of the probe components for the universal probe architecture were as follows: anchor strand − 2 µM, arm strand − 3 µM, quencher strand − 4.5 µM.Concentrations of the probe components for the super universal probe architecture were as follows: anchor strand − 2 µM, arm strand − 3 µM, quencher arm strand − 4.5µM, universal quencher strand − 6.75 µM.
Microarray spotting solution preparation.Microarray spotting solution was prepared by diluting the annealed probes with Tris-HCl buffer spiked with MgCl2 to obtain the following final concentration of the components: 1× Tris-HCl buffer (20 mM Tris, 50 mM KCl), 1.5 mM MgCl2, 0.5 µM of the probe, recalculated to the concentration of the anchor strand.
Microarray printing.Microarrays were printed using a Scienion sciFLEXARRAYER S3 microarray printer equipped with PDC 60 microcapillaries.70% relative humidity was maintained in the printing enclosure during the printing process.After printing the arrays remain in the humidity controlled printing enclosure for 20 minutes, then are stored in a desiccator under reduced pressure for a minimum of 30 minutes to dry.Microarrays were stored at room temperature in a desiccator until final assembly and use.

Extended Data Analysis Description.
Intensity Extraction.All data treatment and visualization was executed in MATLAB.After rescaling the raw intensity of all images, semi-automated masking was performed using the imfindcircles MATLAB function based on a circular Hough transform.This yields a binary matrix for each spot specifying which pixels are included in each region-of-interest (ROI).These masks were used to extract the average intensity of each probe location at each time point,  !() ∀  ∈ 1,2, . . .,  "#$% .
Background and Baseline Subtraction.For each ROI, we constructed a local annular background ROI by extending the microarray spot size by 10 pixels in all directions and removing the microarray ROI.The intensity of this background region,  !(), was obtained for each image and was subtracted from the raw fluorescence for the corresponding spot to remove the influence of fluctuations in illumination intensity and ambient lighting.
The starting intensity of each microarray spot is brighter than the background and varies depending on the probe and the microarray print batch.To remove the influence of this factor, we calculated a 'baseline' intensity for each spot as the average intensity of the spot from images 3 to 7 (i.e. from 1.5 to 3.5 minutes after beginning the run).The baseline does not include the first minute of the run as probes typically alter their starting fluorescence during thermal equilibration and flow stabilization.SNV Identification.Each of the five different SARS-CoV-2 mutation sites (417, 452, 484, 501, and 614) are considered separately.For a given site, the gradient is estimated at all time points beyond the end of the baseline using a fourth-order central difference approximation (note that the ending time point is 29 minutes for this calculation owing to the need for two later images): 12 * 0.5 ∀  ∈ 4, . . .,29 We have found that using a higher-order estimate for the derivative such as this one allows for more accurate SNV calling by limiting the influence of small fluctuations that may be caused by dust particles circulating through the ROI or other local changes that are not removed by our data treatment pipeline.Simpler estimates, such as using the difference in intensity between successive images, are less robust.
Once the slope is estimated for each probe associated with the mutation site in question, the probe with the maximum slope at any point between 4 minutes and 29 minutes is selected as the dominant SNV.As above, all probes associated with this mutation site are normalized as to the maximum fluorescence value across the entire microarray.S6. 484 Probe Sequences.Sequences (5' → 3') used for optimizing probe energetics along the sensitivity-specificity axis.Non-homologous regions (NHRs) are shown in pink and lowercase and are only present in the first three energetic schemes.The double-stranded domain region is in black.The single-stranded toehold region is colored according to the scheme used in Figure 3 (blue for 484E, red for 484K, green for 484Q, gray for 484STOP).The SNV difference between the four variants is in bold.S7. 484 Templates.Sequences (5' → 3') used to test the probes described in Table S6.150

Supplemental Tables
The longest probe-binding region (corresponding to the energetic schemes without any NHRs) 151 is highlighted in bold.The single-SNV differences between the targets are colored according to 152 the scheme in Figure 3 and described in Table S8 S9.Probe Seqs.Sequences (5' → 3') used for detection of all coronaviruses (and influenza A and B) targets.Sequences included in this table only reflect the portions of the viral genome used for detection and do not include proprietary sequences used for anchoring the probe to the surface.

Name
Table S10.Primer Seqs.Sequences (5' → 3') for all primers designed for the respiratory pathogen panel.Forward and reverse primers are designated by fP and rP, respectively, with the reverse primer in all cases responsible both for binding to the template RNA (and thereby mediating cDNA synthesis) and for synthesizing the strand used to displace the quencher arm on the probes.This allows for asymmetric RT-PCR to be used both for the reverse transcription and probe displacement.primers in single-plex to assess the influence of the forward-to-reverse primer ratio (fP:rP) on toehold probe detection.The final forward primer concentration was held constant at 83 nM and the reverse primer concentration was tested at 250 nM, 166 nM, and 83 nM, corresponding to primer ratios of 1:3, 1:2, and 1:1, respectively.An input template concentration of 10,000 molecules of thermally released (at 75 °C for 5 minutes in a Benchmark MyBlock Mini Dry Bath heat block) SARS-CoV-2 Armored RNA Quant from Asuragen was used with standard RT-caPCR master mix composition and reaction conditions (10-minute RT-stage followed by 30minute caPCR stage).We observe similar quencher displacement in response to either 1:3 or 1:2 primer ratios with an expected delay as the overall primer concentration is reduced.The 1:1 ratio produces significantly less sharp caPCR behavior with an additional delay in amplification.

Supplemental Figures
These results match our theoretical predictions, as using equal forward and reverse primer concentrations would produce equal amounts of the two strands of the amplicon.Within the annealing portion of the chamber (where the probes are localized), this translates to a reduced concentration of unbound ssDNA capable of displacing the quencher arm and thus detection kinetics that do not accurately reflect the progress of the reaction (as the displacing strand may also just bind to a free reverse complement in solution instead of finding the probe on the surface).By providing an excess of the primer that produces the displacing strand, there will still be available ssDNA beyond what could be bound by the reverse complement strand.for all four reactions.10-minute and 5-minute RT stages are broadly similar, suggesting that the overall reaction time can likely be reduced by reducing cDNA synthesis.Even without a dedicated RT stage, some reverse transcription still occurs during the PCR stage, as the reverse transcriptase is still active.However, it is likely that completely forgoing the dedicated RT stage will compromise our limit of detection, so an optimal reaction protocol is likely between these two extremes.Even with the longest RT stage, a reaction lacking an RTase does not show any amplification, confirming that there is no effect of DNA contamination.Poor detection from the 484E probe is due to an unaccounted for SNV in the domain region.
pUC57 plasmids and used to test specific detection of SARS-CoV-1 and SARS-CoV-2 in the presence of one or two mutations.The probe-binding region is colored according to the scheme used in Figure3(blue for SARS-CoV-1, red for SARS-CoV-2).Single mutations are highlighted using the opposite color, as the mutations in SARS-CoV-2 templates come from the SARS-CoV-1 sequence and mutations in the SARS-CoV-1 template come from the SARS-CoV-2 sequence.For each target, two single-mutation templates were constructed along with the exact match and a template with both mutations.
Figure S1.Primer Ratio.A set of experiments was performed using only SARS-CoV-1/2

Figure S2 .
Figure S2.SARS-CoV-2 Probe Locations and Microarray Layout.Schematic of probe locations within the SARS-CoV-2 genome.3 amplicons are used to capture 5 common mutation

Figure S3 .
Figure S3.Determining the limit of detection.Limit of detection testing for SARS-CoV-2 and coronavirus 229E in the presence of controls.Titration of viral target RNA with MS2 and IPC

Figure S4 .
Figure S4.Detection of Influenza via RT-caPCR.By introducing three more primer sets targeting the sequence of matrix protein M1 of influenza A and nonstructural protein nsp1 in the Yamagata and Victoria lineages of influenza B, along with corresponding probes, we are able to

Figure S5 .
Figure S5.Different RT Times.Three runs were performed using 100,000 molecules of the SARS-CoV-2 Delta variant RNA and 5,000 molecules of IPC control RNA and varying the time

Figure S6 .
Figure S6.IPC RNA Source does not greatly affect amplification.Prior to optimizing our reaction conditions, we had been using Asuragen's Armored RNA technology as a source of our

Table S1 -
S4. VTM Statistics.Summary information for all VTM samples used for extraction testing.Age is given in years.All other quantities are given as the number falling into each category (out of 50 total VTMs).

Table S5. Panel Product IDs. Product
information used for each template source.All SARS-CoV-2 templates and human coronaviruses 229E and NL63 were obtained from Twist Bioscience, according to the Control number identifier given in column 3.All other templates were synthesized via IVT.The final column gives the reference accession number for each template.These sequences were used for primer and probe design.

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Single mutations used to evaluate probe 153 robustness and taken from the SARS-CoV-2 Delta AY and Omicron spike sequences are 154 denoted using italics.The first four templates have no mutations aside from that specified by the 155 template name and colored accordingly.The next four have one mutation found in both Delta 156 AY and Omicron lineages.The final four have an additional mutation found only in Omicron.